Background-Infantile hemangiomas (IHs) can cause significant morbidity during proliferation, yet there is no FDA-approved treatment. IHs are believed to form from stem cells (HemSCs), which differentiate towards an endothelial cell (HemECs) phenotype. Recently, propranolol has demonstrated effectiveness in the treatment of complicated IHs. We hypothesize that propranolol facilitates IH involution by altering cellular behavior in both HemECs and HemSCs.

Background Infantile hemangiomas are the most common tumor of infancy, yet there are no FDA-approved therapeutics to date. Recently, the non-selective beta-adrenergic-blocker propranolol has been shown to be a safe and effective means of treating infantile hemangiomas, though its mechanism has yet to be elucidated. We have previously demonstrated that propranolol induces early and incomplete adipogenesis in stem cells derived from hemangiomas. We hypothesize that propranolol promotes dysregulated adipogenesis via the improper regulation of adipogenic genes. Methods Hemangioma stem cells isolated from resected infantile hemangioma specimens were treated with adipogenic medium for 1 or 4 days in either propranolol or vehicle. Cell death was measured by the incorporation of annexin V and propidium iodide by flow cytometry. Adipogenesis was assessed by visualizing lipid droplet formation by Oil Red O staining. Pro-adipogenic genes C/EBPβ, C/EBPβ, C/EBPδ, PPARδ, PPARγ, RXRα, and RXRγ were analyzed by quantitative reverse transcription and polymerase chain reaction. Results Hemangioma stem cells treated with propranolol increased lipid droplet formation compared to vehicle-treated cells indicating increased adipogenesis. Cell death as measured by FACS analysis indicated that the propranolol-treated cells died due to necrosis and not apoptosis. During adipogenesis, transcript levels of PPARδ, PPARγ, C/EBPβ, and C/EBPδ were significantly increased (p < 0.01) in propranolol-treated cells relative to control cells. In contrast, RXRα and RXRγ levels were significantly decreased (p < 0.05), and C/EBPα, a gene required for terminal adipocyte differentiation, was strongly suppressed by propranolol when compared to vehicle-treated cells (p < 0.01). Conclusions In hemangioma stem cells, propranolol accelerated dysregulated adipogenic differentiation characterized by improper adipogenic gene expression. Consistent with accelerated adipogenesis, propranolol significantly increased the expression of the pro-adipogenic genes, PPARγ , C/EBPβ and C/EBPγ compared to control. However, propranolol treatment also led to improper induction of PPARδ and suppression of C/EBPα, RXRα and RXRγ. Taken together this data indicates that propranolol promoted dysregulated adipogenesis and inhibited the hemangioma stem cells from becoming functional adipocytes, ultimately resulting in cell death. Understanding this mechanism behind propranolol's effectiveness will be a vital factor in producing more effective therapies in the future.

BackgroundInfantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs.Materials and MethodsHemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR.ResultsHEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes.ConclusionsHES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.

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78 lipoproteoplex by incorporating varying CSP:siRNA ratios into a standardized CLN and treated 3T3 fibroblasts to assess gene expression changes. Cellular internalization of fluorescent siRNA (siGLO) packaged in lipoproteoplex was visualized with fluorescent microscopy. In vivo, we applied lipoproteoplex with siGLO or siRNA to the skin of C57BL/6 mice. We then measured biodistribution via IVIS imaging, dermal penetration using histological sections, and gene expression in treated skin and internal organs 0-5 days post-treatment.

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PurPose: Infantile hemangiomas (IHs) are vascular hyperplasias with high flow and have been proposed to originate from hemangioma stem cells (HemSCs). Proper vessel maturation requires interactions between endothelial cells and their surrounding mural cells. IH pathology has been proposed to be partially due to defective mural cell function. Previous studies in our lab showed NOTCH3 inhibition in HemSC resulted in reduced blood flow in a murine IH model as detected by US Doppler. This correlated with reduced vessel diameter when compared to controls. Since Notch3 functions to regulate mural cell maturation, we determined if Notch3 has a role in mural cell differentiation of HemSCs.Methods: CD133+ HemSCs were isolated from resected hemangioma specimens. HemSC were transduced with lentivirus encoding a NOTCH3 shRNA (HemSC-shN3), an activated form of NOTCH3 (HemSC-N3IC) or a virus containing a scrambled sequence (HemSC-scr) serving as a control. HemSC-N3IC, HemSC-shN3 and HemSC-scr were grown in mural cell differentiation media. After two weeks, immunofluorescent staining was performed with antibodies against the mural cell markers, neuron-glial antigen 2 (NG2) and alpha smooth muscle actin (α-SMA). Fluorescence was quantified and averaged over multiple areas with ImageJ. results: All CD133+ HemSC cell lines expressed low levels of NG2 at baseline (data not shown). When mural cell differentiation was induced, NOTCH3 knockdown upregulated NG2 compared to HemSC-scr. In contrast, NOTCH3 activation decreased NG2 expression. Expression of α-SMA was strongly upregulated in HemSC-scr and Hem-N3IC to a similar level in differentiation medium compared to cell maintained in growth medium (basal). α-SMA expression was unchanged from basal conditions in HemSC-shN3 cells. ConClusions:In HemSCs, perturbations in NOTCH3 level differential altered the expression of mural cell proteins NG2 and α-SMA. In mural cell differentiation conditions NOTCH3 activation strongly downregulated NG2, while having no effect on the expression of α-SMA. In contrast, NOTCH3 inhibition modestly increased NG2 levels, and suppressed the upregulation of α-SMA. Thus, precise regulation of NOTCH3 signaling levels maybe necessary for proper mural cell differentiation of HemSCs.PurPose: Propranolol has shown efficacy in the treatment of problematic infantile hemangiomas (IHs). Propranolol achieves this in part via an anti-vasculogenic effect on hemangioma endothelial cells. Previous studies in our laboratory suggested that propranolol induces accelerated adipogenesis of hemangioma stem cells (HemSCs) in culture. The goal of this study is to investigate the effects of propranolol treatment in a murine model of IH. We hypothesize that systemic propranolol will accelerate involution by affecting both vasculogenesis and adipogenesis.Methods: HemSCs isolated from human specimens and suspended in matrigel were injected subcutaneously into immunodeficient mice. Twenty-four hours after injection, mice were continuously administered oral propranolol or vehicle solutio...

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